Abstract Tuberculosis (TB) is the world?s leading infectious disease threat, causing over a million deaths per year. An effective vaccine is urgently needed to turn the tide against this pandemic. In this project, we propose to develop a live-attenuated whole cell vaccine for TB, as has been done successfully for a range of other pathogens. The TB vaccine currently in use worldwide, BCG, has widely variable efficacy. BCG provides evidence that vaccine generated immune protection against TB is possible, while also making clear that a more effective vaccine is needed to impact the global burden of TB. Based on our preliminary work, we propose to develop a conditionally replicating Mtb strains that could be used as a vaccine for TB. Our strategy to develop candidate strains has several key features: to mimic virulent Mtb in terms of antigenicity, to allow replication of the vaccine strain for a defined number of generations in order to elicit a protective immune response, and to ensure rapid and complete killing of the vaccine strain. Vaccine strains will be developed using novel systems developed in our labs that control mycobacterial growth and death. Duration of vaccine strain replication after vaccination that that produces a protective immune response will be empirically determined. Candidate vaccine strains will be assayed for safety and efficacy of protection against challenge with virulent Mtb in the mouse model of TB. Ultimately, the lead candidate vaccine strain will be tested for safety and efficacy of protection in the cynomolgus macaque model of TB. If these studies identify a successful candidate vaccine, we will work with an industry partner consultant and our industry partner, Ambrx, to bring this product to human trials, including navigating the FDA regulatory path and addressing several issues in the manufacturing of the lead candidate vaccine strain, in order to produce a strain that would be acceptable for use in humans. This project will lead to a deeper understanding of what is required for a protective immunity in the most widely used animal model of TB (mouse), as well as in the animal model that is most relevant to human disease (cynomolgus macaque). Ultimately, the project may result in a candidate live-attenuated whole cell vaccine for TB that will be ready to move into further pre-clinical development.